MDCK suspension cell lines in serum-free, chemically-defined media for vaccine production

ABSTRACT

Disclosed is an adapted Madin-Darby canine kidney cell line capable of suspension culture in the absence of serum, and a chemically-defined medium for culture of the adapted MDCK cell line. Further disclosed are culture methods for growing the adapted MDCK cell line and methods for producing a vaccine from the adapted MDCK cell line grown in the chemically-defined medium.

BACKGROUND Field

The present invention relates generally to the field of vaccine virusproduction, and particularly to growth and suspension culture ofMadin-Darby canine kidney (MDCK) cell lines in serum-free,chemically-defined medium.

Description of the Related Art

Currently, vaccines are produced using a variety of methods, includingculture in chicken eggs, primary cells, or cell lines. These methodsinvolve numerous problems with respect to costs, risks, and ability tomaintain large-scale, industrial production levels.

Madin-Darby canine kidney (MDCK) cells are one cell line used forvaccine production, particularly for influenza vaccines. Cell culturefor vaccine production is advantageous over chicken eggs because cellculture does not rely on the availability of eggs and can be performedon a larger scale. However, there are several drawbacks to the cultureof MDCK cells. MDCK cells require undefined supplement, such as serumfor culture, which is expensive and undesirable when makingpharmaceutical products for human use. MDCK cells also require frequentmedia changes, further increasing cost. Furthermore, MDCK cells grow inadherent culture, either on the culture vessel, which requiressignificant surface area; or on microcarriers, which are not costeffective, are labor intensive, and pose a risk of damaging the cells.

U.S. Pat. Nos. 6,825,036 and 8,846,932; and U.S. Patent ApplicationPublication No. 2013/0183741 describe methods and MDCK cell lines forserum-free suspension culture and vaccine production. However, growth ofthese cells in serum-free culture medium required hydrolysate or otheranimal-derived components, which is an undefined media component thatrepresents a disadvantage in cost and controllability.

Accordingly, there remains a need for MDCK cell lines capable ofsuspension culture in serum-free and chemically-defined media forefficient vaccine production.

SUMMARY OF THE INVENTION

The technology described herein relates to adapted MDCK cells andculture medium for growing the cells. The technology further relates tomethods of culturing the cells and methods for producing vaccines usingthe adapted cells. The adapted MDCK cells as described herein have beenadapted to serum-free suspension culture without the use ofmicrocarriers. The adapted MDCK cells are further cultured in thepresence of a chemically-defined medium as described herein.

The MDCK cell line is an adherent cell line derived from the kidney of anormal female adult Cocker Spaniel. MDCK cells are used for virusproduction, e.g. to produce vaccines. Currently, MDCK cells are culturedin serum-containing medium as adherent cultures. In order to grow MDCKcells to a density that is suitable for commercial production,microcarriers (e.g., microbeads) or roller-bottles are used to increasethe surface area for MDCK cell growth. Microcarrier or roller-bottleculture of MDCK has numerous drawbacks, including increased expense andlimitation of scalability.

Cell lines, including MDCK cells, are conventionally grown inserum-containing media to aid growth and proliferation of the cells.However, serum is not advantageous, particularly in large-scalecommercial production, due to its high cost, risk of contamination,undefined nature, and variability. Such disadvantages spurred the use ofserum-free medium for culturing some cell lines. However, serum-freemedium still contains animal components, e.g. transferrin, and otherundefined products, e.g. hydrolysate, which provide a level of risk anduncertainty when using such medium.

Chemically-defined medium, which contains only recombinant proteinsand/or hormones and for which the precise medium constituents andconcentrations are known, provides an improved system for growth of celllines used in commercial production of viruses for vaccines. Chemicallydefined medium does not contain any animal-derived ingredients.

The ability of a given cell line to grow in serum free,chemically-defined medium is unpredictable. The adaptation of a cellline to suspension growth, particularly in a given medium, is similarlyunpredictable. For example, cells may die off when they are grown insuspension culture without the opportunity to adhere to a surface; cellsmay die off when serum is removed from the medium; chemically definedmedium may not be sufficient to sustain growth of the cells; cell growthand/or proliferation may be negatively affected (e.g., by lengtheningdoubling time); cell morphology and/or gene expression may be alteredsuch that the cells are no longer suitable for their intended purpose;characteristics of the cells may be altered such that virus made by thecells is insufficiently antigenic to be used in a vaccine; and so forth.

The disclosure herein is predicated, in part, on the surprisingdiscovery that MDCK cells can be adapted to suspension growth in serumfree, chemically-defined medium, and that the adapted MDCK cells producehigher titers of virus for vaccine production than the parental cellline, which is adherent and cultured in the presence of serum, and theviruses produced by the adapted cells maintain antigenicity.

In one aspect, this disclosure relates to a composition comprisingadapted MDCK cells and a growth medium, the growth medium comprising achemically-defined and animal component free medium, wherein the adaptedMDCK cells are maintained in suspension culture without microcarriers.

In one aspect, this disclosure relates to a method for culturing anadapted MDCK cell line in suspension without microcarriers, the methodcomprising contacting the adapted MDCK cell line with achemically-defined and animal component free growth medium, wherein theadapted MDCK cell line is capable of growing in suspension culturewithout microcarriers. In one embodiment, the cells are grown in thepresence of 5% CO₂. In a preferred embodiment, the medium is notexchanged during cell culture stage.

In one aspect, this disclosure relates to a method for producing avaccine in an adapted MDCK cell line, said MDCK cell line being capableof being cultured in suspension without microcarriers, said methodcomprising contacting the MDCK cell line with a chemically-defined andanimal component free growth medium. In one embodiment, MDCK cells areinoculated at a concentration of 2×10⁵ cells/ml and virus infection isconducted when the cells approaching stationary phase, as the celldensity reaches about 2×10⁶ cells/mL. In one embodiment, before virusinfection, media is 100% refreshed by centrifugation, and then tosylphenylalanyl chloromethyl ketone (TPCK)-trypsin and strain virus areadded. In one embodiment, during virus infection stage, cells areincubated at 32-34° C. In one embodiment, the cells are grown in spinnerflasks. In one embodiment, the supernatants are harvested when total CPEoccurs. In one embodiment, additional glucose is not added to themedium. In one embodiment, the medium contains about 20 mM to about 30mM glucose, and preferably about 25 mM to about 30 mM glucose, and morepreferably about 26 mM to about 28 mM glucose.

In one aspect, this disclosure relates to a method for producing anadapted MDCK cell line that is capable of being cultured in suspensionwithout microcarriers in a chemically-defined medium without serum, saidmethod comprising:

-   -   a) providing an adherent MDCK cell line;    -   b) culturing the adherent MDCK cell line in a growth medium        comprising 5% (volume/volume, v/v) serum with agitation and in        the absence of microcarriers for a period of time sufficient for        the MDCK cell line to adapt to suspension culture; and    -   c) culturing the suspension-adapted MDCK cell line in suspension        culture in a chemically-defined medium without serum, thereby        providing an adapted MDCK cell line that is capable of being        cultured in suspension without microcarriers in a        chemically-defined medium without serum.

In one embodiment, step c) includes: culturing the suspension-adaptedMDCK cell line in suspension culture with an increasing amount of achemically-defined medium and a decreasing amount of serum (v/v) untilthe chemically-defined medium represents substantially all of the mediumin the culture and substantially all of the serum has been removed,thereby providing an adapted MDCK cell line that is capable of beingcultured in suspension without microcarriers in a chemically-definedmedium without serum.

In one embodiment, the adapted MDCK cell line is cultured in thepresence of 5% CO₂ in steps b) and c).

In certain embodiments, the adapted MDCK cells have an average doublingtime of between about 30 hours and about 35 hours.

In certain embodiments, the adapted MDCK cells are suitable forproducing virus for a human vaccine. In certain embodiments, the virusproduced by the adapted MDCK cells is antigenic. In preferredembodiments, the virus is an influenza virus.

In certain embodiments, the adapted MDCK cells are the cells asdeposited with Leibniz-Institut DSMZ-Deutsche Sammlung vonMikro-organismen and Zellkulturen GmbH (InhoffenstraBe 7 B, 3 8124Braunschweig, Germany) as deposit number DSM ACC3309 on Oct. 21, 2016.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an overview of the protocol used to adapt the MDCK cells forsuspension culture in chemically-defined, serum-free media.

FIG. 2A is photomicrograph of adherent MDCK (aMDCK) cells attached tomicrocarriers. Cells were cultured in serum-free medium.

FIG. 2B is photomicrograph of adapted MDCK (sMDCK) cells grown withoutmicrocarriers. Cells were cultured in chemically-defined, animalcomponent-free medium.

FIG. 3 shows the viable cell density over time of cultures of aMDCKcells (♦) versus sMDCK cells (▴).

FIG. 4 shows the results of a sMDCK cell growth test over ten passages.

FIG. 5 shows the results of a H7N9 flu virus production test performedduring several passages (3^(rd) through 10^(th)) of the sMDCK cells.

FIG. 6 shows the results of a H5N1 flu virus production test performedduring several passages (3^(rd) through 10^(th)) of the sMDCK cells.

DETAILED DESCRIPTION

After reading this description it will become apparent to one skilled inthe art how to implement the invention in various alternativeembodiments and alternative applications. However, all the variousembodiments of the present invention will not be described herein. Itwill be understood that the embodiments presented here are presented byway of an example only, and not limitation. As such, this detaileddescription of various alternative embodiments should not be construedto limit the scope or breadth of the present invention as set forthbelow.

Before the present invention is disclosed and described, it is to beunderstood that the aspects described below are not limited to specificcompositions, methods of preparing such compositions, or uses thereof assuch may, of course, vary. It is also to be understood that theterminology used herein is for the purpose of describing particularaspects only and is not intended to be limiting.

The detailed description of the invention is divided into varioussections only for the reader's convenience and disclosure found in anysection may be combined with that in another section. Unless definedotherwise, all technical and scientific terms used herein have the samemeaning as commonly understood by one of ordinary skill in the art towhich this invention belongs.

In this specification and in the claims that follow, reference will bemade to a number of terms that shall be defined to have the followingmeanings:

The terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting of the invention. Asused herein, the singular forms “a”, “an” and “the” are intended toinclude the plural forms as well, unless the context clearly indicatesotherwise.

“Optional” or “optionally” means that the subsequently described eventor circumstance can or cannot occur, and that the description includesinstances where the event or circumstance occurs and instances where itdoes not.

The term “comprising” is intended to mean that the compositions andmethods include the recited elements, but not excluding others.“Consisting essentially of” when used to define compositions andmethods, shall mean excluding other elements of any essentialsignificance to the combination. For example, a composition consistingessentially of the elements as defined herein would not exclude otherelements that do not materially affect the basic and novelcharacteristic(s) of the claimed invention. “Consisting of” shall meanexcluding more than trace amount of other ingredients and substantialmethod steps recited. Embodiments defined by each of these transitionterms are within the scope of this invention.

The term “about” when used before a numerical designation, e.g.,temperature, time, amount, concentration, and such other, including arange, indicates approximations which may vary by (+) or (−) 10%, 5% or1%.

As used herein, the term “Madin-Darby canine kidney cells” or “MDCKcells” refers to cells from a cell line derived from the tissue of anapparently normal adult female cocker spaniel in 1958. MDCK cells areoften used for vaccine production, particularly influenza. MDCK cellsare commercially available, for example from BCRC (catalogue no. 60004,derived from ATCC CCL-34). Commercially available MDCK cells areadherent in culture and require serum for optimal growth.

As used herein, the term “adapted MDCK cells” refers to cells that havebeen adapted to grow in suspension culture (without microcarriers) inchemically-defined medium, without serum.

As used herein, the term “chemically defined medium” refers to cellculture medium in which all of the components are known, as are theirexact concentrations. Chemically defined medium contains noanimal-derived components.

As used herein, the term “adherent culture” refers to cell culturewherein the cells adhere to a surface, e.g. a tissue culture plate ormicrocarrier. “Microcarriers” are any carrier, e.g. beads, that providesa surface for cells to adhere other than the surface of the culturevessel.

As used herein, the term “suspension culture” refers to cell culture insuspension, as opposed to adherent culture, without the use ofmicrocarriers.

As used herein, the term “substantially all,” for example when referringto the reduction of serum and/or growth medium in a culture, means thatthe undesired component comprises less than about 0.1% of the culturemedium, and preferably less than about 0.05%, and more preferably lessthan about 0.01%. In a most preferred embodiment, all of the undesiredcomponent is removed from the culture medium. Similarly, the desiredcomponent comprises more than about 99.90%, preferably more than about99.95%, and more preferably more than about 99.99%. In a most preferredembodiment, the desired component (e.g., chemically defined medium)comprises 100% of the medium.

Compositions

The present disclosure relates to compositions comprising adapted MDCKcells and a chemically-defined growth medium. The composition does notcomprise serum or other animal-derived components.

Adapted MDCK Cell Lines

In one aspect, the present disclosure relates to adapted MDCK cells thatare capable of growth and/or proliferation without serum inchemically-defined medium in suspension culture. The adapted MDCK cellsdo not require a surface (e.g. microcarriers) for growth orproliferation in suspension culture. The cells can be used to producevirus for vaccine production.

In some embodiments, the adapted MDCK cells have an average doublingtime of between about 20 hours and about 40 hours. In some embodiments,the adapted MDCK cells have an average doubling time of between about 25hours and about 40 hours. In some embodiments, the adapted MDCK cellshave an average doubling time of between about 30 hours and about 40hours. In some embodiments, the adapted MDCK cells have an averagedoubling time of between about 35 hours and about 40 hours. In someembodiments, the adapted MDCK cells have an average doubling time ofbetween about 20 hours and about 35 hours. In some embodiments, theadapted MDCK cells have an average doubling time of between about 20hours and about 30 hours. In a preferred embodiment, the adapted MDCKcells have an average doubling time of between about 30 hours and about35 hours.

In some preferred embodiments, the adapted MDCK cells are suitable forproducing virus for a human vaccine. Viruses that can be produced in theadapted MDCK cells include, without limitation, A/Vietnam/1194/04 (H5N1)virus (NIBRG-14) and A/Anhui/1/2013 (H7N9) virus (NIBRG-268) are used asexamples. In an especially preferred embodiment, the virus is aninfluenza virus. In an especially preferred embodiment, the vaccine is ahuman vaccine.

In one embodiment, the virus produced by the adapted MDCK cells retainsantigenicity. In one embodiment, the virus is capable of causing animmune reaction in a host cell or organism. In a preferred embodiment,the host cell or organism is a human cell or a human.

In certain embodiments, the adapted MOCK cells are the cells asdeposited with Leibniz-Institut DSMZ-Deutsche Sammlung vonMikro-organismen and Zellkulturen GmbH as deposit number DSM ACC3309 onOct. 21, 2016. The cells were submitted for deposit on Oct. 19, 2016.

The chemically-defined medium can be any chemically-defined medium thatdoes not contain serum, hydrolysates, or other animal-derivedcomponents. In a preferred embodiment, the chemically-defined medium isBALANCD® Simple MDCK (Irvine Scientific, catalog ID: 91136). In someembodiments, the chemically-defined medium comprises one or more of theamino acids as set forth in Table 1. In some embodiments, the aminoacid(s) are present in the medium at a concentration within the range(s)set forth in Table 1. The concentration may be any subrange or valuewithin a given range, including endpoints. In some embodiments, one ormore amino acids is present in the medium at the preferred concentrationprovided in Table 1.

TABLE 1 Example Amino Acid Constituents of Chemically-Defined MediumConc. Range Preferred Conc. Description (mM) (mM) L-SERINE  0.1-5 1.1114L-ARGININE HCl  0.1-5 1.1193 L-LEUCINE 0.05-4 0.7192 L-TYROSINE 2Na•2H2O0.05-4 0.3419 L-ISOLEUCINE 0.05-4 0.6632 L-THREONINE 0.05-4 0.7194L-VALINE 0.05-4 0.7196 L-CYSTEINE HCl•H2O 0.05-4 0.4555 L-Aspartic Acid0.05-4 0.5770 L-Glutamic Acid 0.05-4 0.4894 L-ASPARAGINE 0.05-4 0.4050L-PHENYLALANINE 0.05-4 0.3437 L-HISTIDINE HCl H2O 0.02-2 0.2403L-Methionine 0.02-2 0.1848 L-ALANINE 0.02-2 0.2694 L-TRYPTOPHAN 0.02-20.0707

In some embodiments, the chemically-defined medium comprises glucose. Insome embodiments, the concentration of glucose is between about 20 mMand about 30 mM. In a preferred embodiment, glucose is present at aconcentration of about 25 mM to about 30 mM. In an especially preferredembodiment, glucose is present at a concentration of about 27.75 mM. Theconcentration may be any range or value within the ranges recitedherein, including endpoints.

Methods of Making and Culturing Suspension-Adapted MDCK Cells

The present disclosure relates to methods of making and/or culturingMDCK cells/cell line in a chemically-defined, serum-free medium. TheMDCK cells are cultured in suspension and do not require adhesion to aculture vessel or microcarriers.

Making Suspension Adapted MDCK Cell Lines

In one aspect, the current disclosure relates to a method for making asuspension-adapted MDCK cell line (MDCK cells) that is capable of growthand/or proliferation in a chemically-defined medium.

In some embodiments, this disclosure relates to method for producingadapted MDCK cells (cell line) that is capable of being cultured insuspension without microcarriers in a chemically-defined medium withoutserum, said method comprising:

-   -   a) providing adherent MDCK cells;    -   b) culturing the adherent MDCK cells in a growth medium        comprising serum and in the absence of microcarriers for a        period of time sufficient for the MDCK cells to adapt to        suspension culture to produce suspension-adapted MDCK cells; and    -   c) culturing the suspension-adapted MDCK cells in suspension        culture in a chemically-defined medium without serum, thereby        providing MDCK cells that are capable of being cultured in        suspension without microcarriers in a chemically-defined medium.

In one embodiment, the growth medium of step b) is not achemically-defined medium. In one embodiment, the chemically-definedmedium is BALANCD® Simple MDCK medium.

In one embodiment, the cells are grown in a stirred bioreactor.

In one embodiment, step c) includes: culturing the suspension-adaptedMDCK cell line in suspension culture with an increasing amount of achemically-defined medium and a decreasing amount of serum (v/v) untilthe chemically-defined medium represents substantially all of the mediumin the culture and substantially all of the serum has been removed,thereby providing an adapted MDCK cell line that is capable of beingcultured in suspension without microcarriers in a chemically-definedmedium without serum.

In one embodiment, the MDCK cells are cultured in about 1% to about 20%serum in step b). In one embodiment, the MDCK cells are cultured inabout 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%,about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about14%, about 15%, about 16%, about 17%, about 18%, about 19%, or about 20%serum in step b). Ranges include any ranges or values between any twovalues recited herein, including sub-ranges. In a preferred embodiment,the MDCK cells are cultured in about 2% to about 10% serum in step b).In an especially preferred embodiment, the MDCK cells are cultured inabout 2% to about 5% serum in step b).

In one embodiment, step c) involves removing at least a portion of themedium from the cell culture and replacing the removed medium withchemically-defined medium. In one embodiment, step c) involvesseparating MDCK cells from the removed medium and transferring theseparated cells back to the MDCK culture. In one embodiment, step c)involves increasing the portion of chemically defined medium withpassages. In one embodiment, step c) involves directly adapting thecells into serum-free, chemically defined medium.

In a preferred embodiment, the serum-free, chemically-defined medium isBALANCD® Simple MDCK medium.

Culturing Suspension-Adapted MDCK Cell Lines

In one aspect, the current disclosure relates to a method for culturingMadin-Darby canine kidney (MDCK) cells in suspension withoutmicrocarriers. In one embodiment, the method comprises contacting thesuspension-adapted MDCK cells with a chemically-defined medium. Inpreferred embodiments, no serum or other animal-derived components arepresent in the growth medium. In one embodiment, the cells are grown at5% CO₂. In one embodiment, the cells are passaged approximately every3-5 days. Preferably, the cells are passaged approximately every 4 days.In an especially preferred embodiment, the medium is not changed duringculture (e.g., once the cells have been established in culture).

In preferred embodiments, the suspension-adapted MDCK cells are grownunder conditions that maintain the cells in suspension (e.g. agitation).Such conditions are known in the art. Non-limiting examples includesingle-use stirred-tank reactor, wave bioreactor, spinner flasks, andshaking flasks.

Methods of Preparing Vaccines

When virus productivity from MDCK cells is optimized, it isunpredictable whether the virus produced from the cells will maintainsimilar antigenicity to the virus produced by unmodified cells orprotocols. The present disclosure relates to methods of producing virusfor vaccines using the compositions and culture methods describedherein. In preferred embodiments, the antigenicity of the virus ismaintained. In one embodiment, the disclosure relates to a method forproducing a vaccine in MDCK cells, said MDCK cells being capable ofbeing cultured in suspension without microcarriers and without serum. Ina preferred embodiment, the cells are cultured in a chemically-definedmedium. In one embodiment, the chemically-defined medium is BALANCD®Simple MDCK medium.

In some embodiments, additional glucose is not added to the mediumduring vaccine production.

In some embodiments, the medium is not exchanged during the cell culturestage (e.g., after the cells are established in culture). Currently, atleast a portion of the growth medium of MDCK cells used for during thecell culture stage is removed and replaced daily during the cell culturestage Eliminating this step offers significant benefits, including areduction in the amount of medium used (and over-all cost of theprocess), reduction in the processing steps, etc.

In some embodiments, antigenicity of the virus produced by the MDCKcells is evaluated. In preferred embodiments, the virus maintainsantigenicity. In some embodiments, the virus maintains antigenicityrelative to virus produced by adherent MDCK cells. In some embodiments,the virus maintains antigenicity with respect to the requiredantigenicity for production of the desired vaccine. In some embodiments,the virus maintains antigenicity with respect to cells or an organism.In some embodiments, the cells are human cells. In some embodiments, theorganism is a human.

“Maintaining antigenicity” refers to the ability of the virus (orportion of a virus) produced by the MDCK cells to induce an immuneresponse, e.g. in a cell or an organism. Antigenicity can be measured byany method, e.g. HI assay.

EXAMPLES

Additional embodiments are disclosed in further detail in the followingexamples, which are not in any way intended to limit the scope of theclaims.

Example 1: Adaptation of MDCK Cells to Suspension Culture inChemically-Defined, Serum-Free Medium

A schematic of one, non-limiting example protocol for adaptation ofadherent MDCK cells to serum-free suspension culture is provided in FIG.1 . Briefly, adherent MDCK cells (aMDCK) (1) were serially adapted intosuspension culture using medium containing 5% (v/v) fetal bovine serum(FBS) (2). Suspension-adapted MDCK cells (sMDCK) were then adapted intoserum-free BALANCD® Simple MDCK medium (Irvine Scientific) (3). aMDCKcells, grown on CYTODEX™ 1 beads (GE Life Sciences), were also adaptedinto serum-free medium, without adaptation to suspension culture. Virusproductivity and doubling time is provided for each condition.

Prior to adaptation of the aMDCK cells into suspension culture, MDCKcells were initiated as adherent cultures from a frozen working cellbank of cells growing in Dulbecco's Modified Eagle Medium (DMEM) with 5%FBS. The cells were passaged several times in T-flasks and serialadapted into medium containing 5% FBS. That is, the concentration ofDMEM in the growth medium was slowly decreased and the concentration ofBALANCD® Simple MDCK medium was slowly increased over time untilsubstantially all of the growth medium in the T-flask comprised BALANCD®Simple MDCK medium.

Cells in the T-flasks were trypsinized using Trypsin-EDTA, and then thecells were centrifuged at 1000 rpm (200 g) to remove the trypsin. Thecells were resuspended in fresh medium containing 5% FBS and were seededin 125 mL spinner flasks at 5×10⁵ cells/mL in a total volume of 60 mL.Spinner flasks were placed on a stir plate (45˜55 rpm) in a 37° C.,humidified incubator with 5% CO₂. MDCK suspension cultures wererefreshed with 33% BALANCD® Simple MDCK medium containing 5% FBS every3-4 days. The cell density declined to approximately 1×10⁵ to 2×10⁵cells/mL within one week, and the viability was above 50%. MDCK cellsstarted to grow at the second or third week. Doubling times in MDCKsuspension cultures were similar to those seen in adherent microcarriercultures (30-40 hours) and the cells grew in single-cell suspension(i.e. minimal aggregation). Maximum MDCK cell densities in suspensioncultures are approximately 2×10⁶ cells/mL with viabilities >90%. Thesesuspension-adapted MDCK cells were frozen in serum-free medium with 10%DMSO as a master cell bank.

The suspension-adapted MDCK cells from the frozen master cell bank werethawed and directly initiated as suspension cultures in BALANCD® SimpleMDCK medium containing 5% FBS. At the second passage after thawing, thecells were directly adapted into completely serum-free BALANCD® SimpleMDCK medium. At the third passage after thawing, the cells were frozenin serum-free medium with 10% DMSO to create a working cell bank.

The suspension-adapted, serum free-adapted MDCK cells (sMDCK) from thefrozen working cell bank were thawed and directly initiated assuspension cultures in BALANCD® Simple MDCK, without serum. MaximumsMDCK cell densities in suspension cultures were approximately 2×10⁶cells/mL with viabilities >90%. Doubling time for sMDCK was 30-40 hours.Cells were grown in spinner culture at approximately 50 to 70 rpm in 5%CO₂.

Example 2: Aggregation of MDCK Cells

FIG. 2A shows a photo micrograph of aMDCK cells attached to Cytodex1beads in serum-free culture. FIG. 2B shows a photo micrograph of sMDCKcells in free suspension culture in serum-free BALANCD® Simple MDCK.

This level of aggregation was obtained without the use of pipetting orother methods of mechanically dispersing the cells (other than the 50rpm spinner speed required for culture of the cells). Without beingbound by theory, it is believed that reducing cell aggregation providesa greater exposed cell surface area and allows for better infection ofthe cells, thereby resulting in a higher viral titer.

Example 3: MDCK Cell Growth Comparison

Growth rates of MDCK cells were evaluated. MDCK cells were seeded into125 mL spinner flasks at a density of 0.2×10⁶ cells/mL to 0.25×10⁶cells/mL and grown in an incubator at 37° C. and 5% CO₂ at a spinnerspeed of 50 rotations per minute (rpm). Cells were passaged every fourdays. sMDCK cells were cultured in BALANCD® Simple MDCK medium (IrvineScientific) supplemented with 4 mM L-glutamine, without microcarriers.aMDCK cells were grown in OPTIPRO™ SFM (Life Technologies) supplementedwith 4 mM L-glutamine, attached to 5 g/L CYTODEX® 1 beads (GEHealthcare). Starting on day 2 of culture, 70% of the culture mediumfrom the aMDCK cells was removed and replaced with fresh medium. Nomedia exchange was performed for sMDCK cells.

FIG. 3 shows the growth rates of the two cell lines over twelve days ofculture. Viable cell density reached ˜2×10⁶ cells/mL in 4 days, asimilar growth pattern compared to the microcarrier culture. Nosignificant change in growth rate was observed.

No medium exchange was necessary for the suspension culture. Incontrast, microcarrier culture required a daily media change of 70%,starting on day 2 of culture to reach high cell density.

Example 4: Influenza Virus Production in sMDCK Cells

The ability of the sMDCK cells to produce virus was determined. MDCKcells were seeded into 125 mL spinner flasks at a density of 0.2×10⁶cells/mL to 0.25×10⁶ cells/mL and grown as described in Example 3. Noadditional glucose was added to the medium.

Once the cells reached a density of 2×10⁶ cells/mL, medium was first100% refreshed by centrifugation. TPCK-trypsin was added into culturemedium and then cells were infected with H7N9 influenza virus at lowmultiplicity of infection (MOI). During virus culture stage, spinnerflasks were incubated at 34° C. The supernatants were harvested whentotal cytopathic effect (CPE) occurred.

Maximum sMDCK cell densities in suspension cultures are approximately2×10⁶ cells/mL with viabilities >90%. Doubling times in sMDCK are 30-40hours and the cells grow in aggregated-cell suspension (FIG. 4 ). Foreach sample, two serial 1:2 dilutions (12×100 μl each) were prepared inround bottomed 96-well microtiter plates. Serial dilutions were shiftedby a factor of 1:2^(0.5) resulting in a dilution factor of 1:2^(0.5)from well to well. One hundred microliters of 0.25% turkey erythrocytes(2×10⁷ RBC/mL) were added to each well and plates were incubated at roomtemperature for at least 2 hours up to one day. Afterwards, plates werescanned with a plated photometer measuring extinction at 700 nm. Thetransition from carpet-like sedimentation of erythrocytes (in thepresence of negligible virus) could be detected as an increase inextinction levels. A Boltzmann sigmoid was fitted to each data set andthe dilution at the point of inflection (one of the parameters) wasdefined as the endpoint of the titration; the inverse of the dilutionwas defined as the specific HA activity with units 1 HAU (100 μL)⁻¹. Aninternal standard was used to compensate for fluctuations caused by thevarying quality of turkey erythrocytes. Samples belonging to the sameexperiment were analyzed in the same assay run whenever possible.Statistical analysis of multiply determined samples predicted ananalytical error <15% (confidence interval, alpha=0.05) formeasurements.

The infectious virus titer was measured by determining the tissueculture infective dose required to infect 50% of MDCK cells. Tenfoldserial dilutions (10⁻¹ to 10⁻⁸) of the virus samples were prepared inmedium containing TPCK-trypsin and inoculated (six replicates perdilution) in 96-well plates grown to confluence with MDCK cells. Plateswere incubated at 34° C. for 4 to 7 days. The wells with live cells orwith CPE were calculated in each dilution. The TCID50 titer was furthercalculated by Reed-Muench Method

The results are summarized in Table 2. sMDCK cells provided a highertiter than aMDCK cells.

TABLE 2 H7N9 Production: Peak virus titer during virus propagation stageHA (units/100 μl) TCID₅₀/mL aMDCK microcarrier culture 574.9 ± 113.54 7.6 ± 0.10 sMDCK suspension culture 996.3 ± 113.88 7.91 ± 0.52

aMDCK cells cultured in BALANCD® Simple MDCK medium had a slightlyhigher titer (512.0 HA units/100 μl) than aMDCK cells cultured inOPTIPRO™ SFM, but still significantly less than the sMDCK cells culturedin BALANCD® Simple MDCK medium. Similar results were observed with H5N1influenza virus.

H7N9 production test was performed during the 3^(rd) passage to the10^(th) passage. The virus productivity of sMDCK remained at a highlevel from the 3^(rd) to 10^(th) passages (FIG. 5 ). The average HAtiter was 989.87 HA units/100 μL and the TCID50 titer was 8.6 TCID₅₀/mL(Table 3). Similar results were observed with H5N1 influenza virus (FIG.6 ).

TABLE 3 H7N9 Production The peak virus titer during virus propagationstage HA (units/100 μl) TCID₅₀/mL aMDCK microcarrier culture 612.72 8.6sMDCK suspension culture 989.87 ± 115.89 8.63 ± 0.44

Example 5: Antigenicity Testing

Antigenicity analysis of the influenza viruses from Example 4 wascarried out by hemagglutination (HI) assay using the standard antibodythat was purchased from the National Institute for Biological Standardsand Control (NIBSC). The HI assay started at a serum dilution of 1:40.Each sample was performed in triplicate.

Influenza viruses were diluted to give a preparation with 8 HAU per 50μl. The antibody was several diluted in a V-shaped microtiter plate andthen 25 μl virus samples were added into microplate. After gentleagitation, the plates were incubated for 15 min at room temperature. The50 μL of a 0.5% suspension of Turkey red blood cells were added to eachwell and the plates were left another 30 min before reading. Thereciprocal value of the highest dilution of antibody which completelyinhibited hemagglutination was determined to be the HI titer. Resultsare provided in Table 4.

TABLE 4 HI test Virus strain HI titer Virus strain HI titer NIBRG14 320NIBRG268 640 Spinner flask 320 Spinner flask 640 (the 10^(th) passage(the 10^(th) passage of sMDCK) of sMDCK)

It will be understood by those of skill in the art that numerous andvarious modifications can be made without departing from the spirit ofthe present disclosure. Therefore, it should be clearly understood thatthe forms disclosed herein are illustrative only and are not intended tolimit the scope of the present disclosure.

What is claimed is:
 1. A cell culture comprising isolated adaptedMadin-Darby canine kidney (MDCK) cells deposited as DSM ACC3309 withLeibniz-Institut DSMZ-Deutsche Sammlung von Mikro-organismen andZellkulturen GmbH and a growth medium that comprises from about 20 mM toabout 30 mM glucose.
 2. The cell culture of claim 1, wherein the growthmedium is a chemically-defined and animal component free medium.
 3. Amethod of producing an influenza virus for vaccine production,comprising contacting isolated adapted Madin-Darby canine kidney (MDCK)cells deposited as DSM ACC3309 with Leibniz-Institut DSMZ-DeutscheSammlung von Mikro-organismen and Zellkulturen GmbH with a growthmedium, infecting the isolated adapted MDCK cells with an influenzavirus, and harvesting the influenza virus.
 4. The method of claim 3,wherein the growth medium is a chemically-defined andanimal-component-free medium.
 5. The method of claim 3, wherein thegrowth medium comprises from about 20 mM to about 30 mM glucose.
 6. Themethod of claim 3, further comprising preparing a vaccine from theharvested influenza virus.
 7. The method of claim 6, wherein the vaccineis a human vaccine.
 8. The method of claim 6, wherein the preparedvaccine comprises a plurality of the harvested influenza viruses.
 9. Themethod of claim 8, wherein the plurality of harvested influenza virusesmaintain antigenicity.